A novel method to detect hPG80 (human circulating progastrin) in the blood.

hPG80 (human circulating progastrin) is produced and released by cancer cells. We recently reported that hPG80 is detected in the blood of patients with cancers from different origins, suggesting its potential utility for cancer detection. To accurately measure hPG80 in the blood of patients, we developed the DxPG80 test, a sandwich Enzyme-Linked Immunosorbent Assay (ELISA). This test quantifies hPG80 in EDTA plasma samples. The analytical performances of the DxPG80 test were evaluated using standard procedures and guidelines specific to ELISA technology. We showed high specificity for hPG80 with no cross-reactivity with human glycine-extended gastrin (hG17-Gly), human carboxy-amidated gastrin (hG17-NH2) or the CTFP (C-Terminus Flanking Peptide) and no interference with various endogenous or exogenous compounds. The test is linear between 0 and 50 pM hPG80 (native or recombinant). We demonstrated a trueness of measurement, an accuracy and a variability of hPG80 quantification with the DxPG80 test below the 20% relative errors as recommended in the guidelines. The limit of detection of hPG80 and the limit of quantification were calculated as 1 pM and 3.3 pM respectively. In conclusion, these results show the strong analytical performance of the DxPG80 test to measure hPG80 in blood samples.

The oncogenic and druggable hPG80 (Progastrin) is overexpressed in multiple cancers and detected in the blood of patients.

BACKGROUND: In colorectal cancer, hPG80 (progastrin) is released from tumor cells, promotes cancer stem cells (CSC) self-renewal and is detected in the blood of patients. Because the gene GAST that encodes hPG80 is a target gene of oncogenic pathways that are activated in many tumor types, we hypothesized that hPG80 could be expressed by tumors from various origins other than colorectal cancers, be a drug target and be detectable in the blood of these patients. METHODS: hPG80 expression was monitored by fluorescent immunohistochemistry and mRNA expression in tumors from various origins. Cancer cell lines were used in sphere forming assay to analyze CSC self-renewal. Blood samples were obtained from 1546 patients with 11 different cancer origins and from two retrospective kinetic studies in patients with peritoneal carcinomatosis or hepatocellular carcinomas. These patients were regularly sampled during treatments and assayed for hPG80. FINDINGS: We showed that hPG80 was present in the 11 tumor types tested. In cell lines originating from these tumor types, hPG80 neutralization decreased significantly CSC self-renewal by 28 to 54%. hPG80 was detected in the blood of patients at significantly higher concentration than in healthy blood donors (median hPG80: 4.88 pM versus 1.05 pM; p < 0.0001) and shown to be correlated to GAST mRNA levels in the matched tumor (i.e., lung cancers, Spearman r = 0.8; p = 0.0023). Furthermore, we showed a strong association between longitudinal hPG80 concentration changes and anti-cancer treatment efficacy in two independent retrospective studies. In the peritoneal carcinomatosis cohort, median hPG80 from inclusion to the post-operative period decreased from 5.36 to 3.00 pM (p < 0.0001, n = 62) and in the hepatocellular carcinoma cohort, median hPG80 from inclusion to remission decreased from 11.54 pM to 1.99 pM (p < 0.0001, n = 63). INTERPRETATION: Because oncogenic hPG80 is expressed in tumor cells from different origins and because circulating hPG80 in the blood is related to the burden/activity of the tumor, it is a promising cancer target for therapy and for disease monitoring. FUNDINGS: ECS-Progastrin.

Targeting the Wnt Pathway and Cancer Stem Cells with Anti-progastrin Humanized Antibodies as a Potential Treatment for K-RAS-Mutated Colorectal Cancer.

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct ß-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS-mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267-80. ©2017 AACR.

Dual Function of NRP1 in Axon Guidance and Subcellular Target Recognition in Cerebellum.

Subcellular target recognition in the CNS is the culmination of a multiple-step program including axon guidance, target recognition, and synaptogenesis. In cerebellum, basket cells (BCs) innervate the soma and axon initial segment (AIS) of Purkinje cells (PCs) to form the pinceau synapse, but the underlying mechanisms remain incompletely understood. Here, we demonstrate that neuropilin-1 (NRP1), a Semaphorin receptor expressed in BCs, controls both axonal guidance and subcellular target recognition. We show that loss of Semaphorin 3A function or specific deletion of NRP1 in BCs alters the stereotyped organization of BC axon and impairs pinceau synapse formation. Further, we identified NRP1 as a trans-synaptic binding partner of the cell adhesion molecule neurofascin-186 (NF186) expressed in the PC AIS during pinceau synapse formation. These findings identify a dual function of NRP1 in both axon guidance and subcellular target recognition in the construction of GABAergic circuitry.

Developmental gene expression profile of axon guidance cues in Purkinje cells during cerebellar circuit formation.

The establishment of precise neural circuits during development involves a variety of contact-mediated and secreted guidance molecules that are expressed in a complementary fashion by different cell types. To build a functional circuit, each cell type must first trigger an intrinsic genetic program that is led by their environment at a key time point. It is therefore essential to identify the different cell-specific and stage-specific transcriptional profiles expressed by neurons. However, very few studies have been done to address this issue thus far. Herein, we have carried out a large-scale quantitative real-time PCR analysis of all classical axon guidance molecules (i.e., Semaphorins, Netrins, Ephrins, and Slits) and their receptors expressed by Purkinje cells (PCs) at specific stages of postnatal cerebellar development in vivo. Most cerebellar connections are setup in a well-characterized sequential manner during postnatal development and lead to the fine regulation of the PC, the sole output of the structure. Our analysis of the relative expression of these guidance cues has uncovered a dynamic expression pattern corresponding to specific stages of cerebellar development, thus providing a starting point for studying the role of these axon guidance molecules in cerebellar wiring.

SEMA3A signaling controls layer-specific interneuron branching in the cerebellum.

BACKGROUND: GABAergic interneurons regulate the balance and dynamics of neural circuits, in part, by elaborating their strategically placed axon branches that innervate specific cellular and subcellular targets. However, the molecular mechanisms that regulate target-directed GABAergic axon branching are not well understood. RESULTS: Here we show that the secreted axon guidance molecule, SEMA3A, expressed locally by Purkinje cells, regulates cerebellar basket cell axon branching through its cognate receptor Neuropilin-1 (NRP1). SEMA3A was specifically localized and enriched in the Purkinje cell layer (PCL). In sema3A(-/-) and nrp1(sema-/sema-) mice lacking SEMA3A-binding domains, basket axon branching in PCL was reduced. We demonstrate that SEMA3A-induced axon branching was dependent on local recruitment of soluble guanylyl cyclase (sGC) to the plasma membrane of basket cells, and sGC subcellular trafficking was regulated by the Src kinase FYN. In fyn-deficient mice, basket axon terminal branching was reduced in PCL, but not in the molecular layer. CONCLUSIONS: These results demonstrate a critical role of local SEMA3A signaling in layer-specific axonal branching, which contributes to target innervation.

Metabolic fingerprints of altered brain growth, osmoregulation and neurotransmission in a Rett syndrome model.

BACKGROUND: Rett syndrome (RS) is the leading cause of profound mental retardation of genetic origin in girls. Since RS is mostly caused by mutations in the MECP2 gene, transgenic animal models such as the Mecp2-deleted ("Mecp2-null") mouse have been employed to study neurological symptoms and brain function. However, an interdisciplinary approach drawing from chemistry, biology and neuroscience is needed to elucidate the mechanistic links between the genotype and phenotype of this genetic disorder. METHODOLOGY/PRINCIPAL FINDINGS: We performed, for the first time, a metabolomic study of brain extracts from Mecp2-null mice by using high-resolution magnetic resonance spectroscopy. A large number of individual water-soluble metabolites and phospholipids were quantified without prior selection for specific metabolic pathways. Results were interpreted in terms of Mecp2 gene deletion, brain cell function and brain morphology. This approach provided a "metabolic window" to brain characteristics in Mecp2-null mice (n = 4), revealing (i) the first metabolic evidence of astrocyte involvement in RS (decreased levels of the astrocyte marker, myo-inositol, vs. wild-type mice; p = 0.034); (ii) reduced choline phospholipid turnover in Mecp2-null vs. wild-type mice, implying a diminished potential of cells to grow, paralleled by globally reduced brain size and perturbed osmoregulation; (iii) alterations of the platelet activating factor (PAF) cycle in Mecp2-null mouse brains, where PAF is a bioactive lipid acting on neuronal growth, glutamate exocytosis and other processes; and (iv) changes in glutamine/glutamate ratios (p = 0.034) in Mecp2-null mouse brains potentially indicating altered neurotransmitter recycling. CONCLUSIONS/SIGNIFICANCE: This study establishes, for the first time, detailed metabolic fingerprints of perturbed brain growth, osmoregulation and neurotransmission in a mouse model of Rett syndrome. Combined with morphological and neurological findings, these results are crucial elements in providing mechanistic links between genotype and phenotype of Rett syndrome. Ultimately, this information can be used to identify novel molecular targets for pharmacological RS treatment.

Brain magnetic resonance study of Mecp2 deletion effects on anatomy and metabolism.

Rett syndrome, a neurodevelopmental X-linked disorder, represents the most important genetic cause of severe mental retardation in the female population and results from a mutation in the gene encoding methyl-CpG-binding protein 2 (MECP2). We report here the first characterization of Mecp2-null mice, by in vivo magnetic resonance imaging and spectroscopy, delineating the cerebral phenotype associated with the lack of Mecp2. We performed a morphometric study that revealed a size reduction of the whole brain and of structures involved in cognitive and motor functions (cerebellum and motor cortex). Significant metabolic anomalies, including reduced N-acetylaspartate, myo-inositol, and glutamine plus glutamate, and increased choline levels were evidenced. These findings indicate that not only neuronal but also glial metabolism is affected in Mecp2-null mice. Furthermore, we uncovered an important reduction of brain ATP level, a hitherto undetected anomaly of energy metabolism that may reflect and contribute to cerebral injury and dysfunction.

Mecp2 deficiency disrupts norepinephrine and respiratory systems in mice.

Rett syndrome is a severe X-linked neurological disorder in which most patients have mutations in the methyl-CpG binding protein 2 (MECP2) gene and suffer from bioaminergic deficiencies and life-threatening breathing disturbances. We used in vivo plethysmography, in vitro electrophysiology, neuropharmacology, immunohistochemistry, and biochemistry to characterize the consequences of the MECP2 mutation on breathing in wild-type (wt) and Mecp2-deficient (Mecp2-/y) mice. At birth, Mecp2-/y mice showed normal breathing and a normal number of medullary neurons that express tyrosine hydroxylase (TH neurons). At approximately 1 month of age, most Mecp2-/y mice showed respiratory cycles of variable duration; meanwhile, their medulla contained a significantly reduced number of TH neurons and norepinephrine (NE) content, even in Mecp2-/y mice that showed a normal breathing pattern. Between 1 and 2 months of age, all unanesthetized Mecp2-/y mice showed breathing disturbances that worsened until fatal respiratory arrest at approximately 2 months of age. During their last week of life, Mecp2-/y mice had a slow and erratic breathing pattern with a highly variable cycle period and frequent apneas. In addition, their medulla had a drastically reduced number of TH neurons, NE content, and serotonin (5-HT) content. In vitro experiments using transverse brainstem slices of mice between 2 and 3 weeks of age revealed that the rhythm produced by the isolated respiratory network was irregular in Mecp2-/y mice but could be stabilized with exogenous NE. We hypothesize that breathing disturbances in Mecp2-/y mice, and probably Rett patients, originate in part from a deficiency in noradrenergic and serotonergic modulation of the medullary respiratory network.